The invention concerns methods for the wavelength calibration of spectrometers. In particular, the invention concerns methods for the wavelength calibration of secondary spectrometers by users where the secondary spectrometers are substantially identical in construction to a primary spectrometer that is available in the factory and serves as a standard for the wavelength calibration. The methods according to the invention can be used for the wavelength calibration of spectrometers which have a detector array as a detection unit and data processing devices for storing and processing data.
Spectrometers are in general devices for displaying a spectrum and enable optical spectra to be recorded and analyzed. Spectrometers are widely used among others in chemical and medical analytics in order to determine components of a liquid on the basis of their spectral properties. Spectrometers that are used for this purpose frequently operate according to the polychromator principle, i.e., in these methods the irradiated light is not split into its spectral components by a polychromator until after it has passed through the sample liquid and the spectral components can thus be imaged simultaneously on a detector array. This enables an entire spectrum to be recorded simultaneously (Optical Multichannel Analyzer (OMA) or Multi Channel Spectrometer (MCS)). This is an advantage over conventional monochromator systems in which the wavelengths have to be scanned successively. Modern multi channel spectrometers can transfer a complete spectrum very rapidly to the evaluation electronics. Typical measuring times are a few milliseconds and typical resolutions of the detector arrays that are used are 128, 256, 512, 1024 or 2048 pixels/spectrum. Other advantages of polychromator spectrometers are the small number of optical components and the omission of mechanically moved parts which makes them considerably cheaper to manufacture.
A typical field of use for spectrometers is for example the analytical determination of hemoglobin derivatives in blood, the so-called CO-oximetry. An example of such a spectrometry module is the COOX module of the cobas b 221 (Roche Diagnostics GmbH, Germany) for determining bilirubin (bili), total hemoglobin (tHb) and the hemoglobin derivatives oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb), carboxyhemoglobin (COHb) and methemoglobin (MetHb). In this case the hemoglobin derivatives and bilirubin are determined on the basis of the Lambert-Beer law. The optical system of this CO oximetry module consists of a halogen lamp, slit, cuvette holder with a cuvette as well as a polychromator and detection unit. The light of a halogen lamp is guided to the cuvette holder with the aid of a light guide. In the cuvette the light is partially absorbed by the sample and partially transmitted. The absorption is characteristic for the composition of the sample. The transmitted light is guided to the polychromator by a further light guide where it is split into its spectral components and imaged on the surface of a photosensitive receiver (CCD sensor). The absorption and ultimately the concentration of the hemoglobin derivatives are calculated from the resulting electrical signal. In order to achieve a high reliability in operation, the polychromator is calibrated with a built-in spectral light source. This calibration is carried out after each switching on of the instrument and at least once daily during the system calibration.
Many analytical applications for spectrometers require instrument-specific calibration data, the generation of which is often time-consuming and costly. For example, apparently identical instruments which are produced by the same manufacturer can exhibit minor instrument variations; such variations are seen when an instrument is built with a component which slightly deviates from the same component in another instrument. Furthermore, a calibrator set for an instrument produced by one manufacturer is in general unsuitable for a similar instrument which is produced by another manufacturer: Furthermore, repairs on a single instrument can have the effect that the spectral response of the instrument varies. If an instrument ages it can change its spectral response. The spectral response of an instrument can additionally vary due to fluctuations in the operating environment. In the case of medical-diagnostic applications which often require an exact analysis of analytes also at very low concentrations, even a small instrument variation can result in an unacceptable error in the analysis.
In the development of analytical instruments for biomedical applications based on spectroscopy there is a need to produce hundreds to many thousands of identically constructed analytical instruments for a certain application. There is no effective method for rapidly and cost-effectively calibrating individual instruments for such large numbers of instruments. Therefore, in this case one endeavors to transfer calibrations from one analytical instrument to the next. This is achieved practically by generating a calibration data set in the factory on a primary spectrometer which serves as a standard. This data set is subsequently transferred to one or many secondary spectrometers that are identical in construction which can then be delivered to the customers together with these calibration data of the primary spectrometer. As mentioned above, even slight individual differences between the primary spectrometer and secondary spectrometer can mean that the calibration data of the primary spectrometer cannot be used without changes on the secondary spectrometer because otherwise this may result in unacceptable inaccuracies in the analyte determination due to the non-instrument-specific calibration data especially in the case of applications for medical diagnostics. A critical calibration parameter of spectroscopic analytical systems is the wavelength calibration, i.e., the assignment of the individual pixels of a recorded spectrum to certain wavelength ranges which are imaged on them.
Such a wavelength calibration can for example be determined by a wavelength assignment in the form of a suitable assignment function which, for example, in the form of a polynomial of the 3rd degree assigns a wavelength λ, for example in nm, to a certain pixel number x by means of the polynomial function λ(x)=a0+a1 x+a2x2+a3 x3.
Various methods are already known in the prior art for calibrating the wavelengths of spectrometers.
In the conventional wavelength calibration methods, a spectrum for example of a neon lamp imaged on the detection unit is used in a peak search method to determine at which pixel the respective emission peaks of the neon lamp, whose exact wavelength is known, are situated. In this case each peak usually extends over several pixels, the maximum can also fall between two pixels. This peak can be represented by a suitable mathematical function using the intensity values of the pixels. One method of determining the position of the peaks as exactly as possible, is an integration of the mathematical function that is used and subsequent subdivision of the area under the curve determined in this manner into two partial areas of equal size where the peak center is defined as that pixel number at which this subdividing line is positioned. In this case it is also possible that the peak center is between two real pixels and thus a non-integral, virtual pixel number can be obtained as a peak center. Those pairs of values consisting of a known wavelength and pixel number of the respective peak must be determined in this manner for each of several peaks which form the design points for a fitting for example to a polynomial of the 3rd degree according to the least squared error method. Typically at least 4 to 8 peaks are required in the peak search method for a sufficiently accurate wavelength calibration. The other measurement ranges of the spectrum in which no sufficiently narrow peaks are to be found, cannot be additionally used in this method. The coefficients determined by the fitting, for example a0, a1, a2, a3 in the case of a fitting to a polynomial of the 3rd degree, at the same time represent the coefficients of a wavelength assignment for example according to λ(x)=a0+a1 x+a2x2+a3 x3.
There are numerous other peak search methods which are used in the prior art. Thus, for example a bandwidth method can be used in which firstly the pixel with the maximum intensity value is determined for one peak. Subsequently the pixel numbers are determined at which a certain percentage, for example 70%, 50% or 30%, of the maximum intensity value is reached. Also in this case interpolation methods can again be used to increase the accuracy and hence non-integral pixel numbers can be obtained as virtual pixel numbers. Two pixel numbers, one to the left and one to the right of the intensity maximum, are obtained from which the sought-after peak center can be obtained by averaging.
Another possible method is the parabola method. The basis of this method is the fact that a parabola can be unequivocally defined by three points. Firstly the pixel of the peak is determined which has the maximum intensity value of the observed peak. In addition the intensity values of the pixels which are to the left and right of this pixel are determined. A parabolic equation can be determined from these three value pairs the apex of which corresponds to the peak center. Also in this case interpolation methods could be used to increase the accuracy so that non-integral pixel numbers can be obtained as virtual pixel numbers.
U.S. Pat. No. 6,700,661 describes a wavelength calibration method in which a defined reference sample is measured on a secondary spectrometer and its (corrected) spectral curve S*sec is compared with a reference spectrum Xsec which is also stored on the instrument. A so-called “spectral residual” value is introduced as a value for the agreement between the two spectra. For the wavelength calibration a wavelength-pixel number relation is used at which a minimum value of the “spectral residual” value is reached. Iterative methods are used to determine this minimum value in order to determine the wavelength-pixel number relation at which the best agreement between the measured spectrum of the reference sample and its deposited reference spectrum occurs.
In order to carry out a wavelength calibration according to this method, it is absolutely essential to measure a reference sample having an exactly defined composition. Additional calibration solutions are required for this, the lot-specific composition of which introduces further variables into the calibration which in turn can impair the accuracy of the calibration. In the case of the iterative method used here, it is additionally very difficult to determine a wavelength assignment with a polynomial of more than the 1st degree which limits the accuracy of the calibration. Furthermore, this method requires the additional step of calculating a correction spectrum which increases the demands on the computing power of a data processing system instructed to carry out the calculation.
Spectrometers are described in U.S. Pat. No. 4,866,644 which use moved optical elements such as swivelling defraction gratings or interference filter arrangements in order to split up the excitation light into its spectral components which are successively irradiated into the sample. Errors in the wavelength assignment can occur in such spectrometer types especially as a result of the mechanical grating drives and controls.
In order to take into account such errors, a wavelength calibration and a spectral correction of the secondary spectrometer with reference to a primary spectrometer are carried out in the factory. For this purpose a large number of different samples are measured on the primary as well as on the secondary spectrometer and specific model spectra are set up on the basis of these data for each sample type. The measured values of the various model spectra are then consolidated into a respective set per wavelength or corresponding unit. A correlation analysis is carried out with about 5 neighboring sets of the secondary spectrum for each set of the primary spectrum. The best correlation value is in this case determined as a maximum value or by fitting the individual values with a quadradic function. This is carried out successively for all sets of the primary spectrum. A new wavelength assignment is determined. by correlation calculations using the function of the 1st degree between the individual value pairs. In this case natural samples such as soy beans or grain are used as samples to carry out a wavelength calibration at the factory. It is therefore difficult for a user to carry out a wavelength calibration at a later time because such natural samples cannot be stored for longer periods without changing their optical properties. The use of such natural reference materials is also critical because it is not possible to ensure a standardization of their spectral properties.
U.S. Pat. No. 5,347,475 describes a wavelength calibration method which is essentially based on monochromatic light which has exactly defined atomic emission lines.
These are used for the purposes of wavelength calibration by determining their known position by a primary and secondary spectrometer. In this case peak search methods are used to find the position of the atomic emission lines which make high demands on the resolution and accuracy of the spectrometers that are used. The methods that are described here are primarily suitable for use in high resolution spectrometers with a resolution of less than 2 nm.
U.S. Pat. No. 5,771,094 describes a method for the continuous monitoring of the wavelength calibration of a secondary spectrometer by determining the position of one or more previously selected peaks or atomic lines from the spectrum of the irradiated light. Conventional peak search methods are used for this purpose. In this manner deviations can be determined from a comparison of the actual values with the peak maxima deposited in the secondary spectrometer and correction measures can be initiated. Also in this case the described methods make high demands on the accuracy and resolution of the spectrometer which must resolve atomic lines.
The wavelength calibration methods described above have the major disadvantage that for system-related reasons they can hitherto only be used for high resolution spectrometers (i.e., spectrometers with the narrowest possible bandwidth) because peaks resolved to a sufficient extent can only be obtained in such a spectrum). FIG. 1 shows in this connection two spectra of the same neon lamp in the wavelength range of 660 nm to 960 nm. The wavelength λ (in nm) is plotted on the x axis and the intensity I (in relative units) normalized to the maximum value is plotted on the y axis. The spectrum shown by a continuous line was recorded with a spectrometer having a bandwidth of 2.5 nm, the spectrum shown with a dashed line was recorded with a spectrometer with a bandwidth of 8 nm.
It can be dearly seen that the narrow peaks of the neon spectrum required for wavelength calibration by means of the peak search method can only be adequately resolved with a high resolution spectrometer in order to ensure an adequate quality of a wavelength calibration. The peaks required for this can no longer be adequately resolved by spectrometers with a lower resolution (see, for example, non-resolved multiple peaks at 660-680 nm or 730-760 nm or shoulders instead of resolved single peaks in the range of 680-700 nm and 870-890 nm) which would greatly reduce the accuracy of a wavelength calibration based on this by means of a peak search method. With the previously described peak search methods, especially those using the surface integration method, a calibration accuracy of ±5·10−3 nm, which is necessary especially for applications in the medical diagnostic field, can only be achieved by using high quality spectrometers of low bandwidth. In this case the calibration accuracy is understood as the maximum error (calculated wavelength minus the reference wavelength) over the entire wavelength range. As a result only high resolution spectrometers have been used for such medical diagnostic analytical systems which, due to their high demands on the quality of manufacture and resolution, are complex and costly. Because, as already mentioned, such analytical systems are often manufactured in large numbers, this restriction to such spectrometers represents a considerable production cost factor.
Hence, for such applications it is desirable to use spectrometers with a smaller overall size and lower price. However, such spectrometers often have poorer optical properties such as a larger bandwidth and asymmetric peak representation. When these spectrometers are calibrated by the user with the aid of a conventional peak search method, calibration accuracies of only up to 1 nm are possible which is not sufficient for applications in medical diagnostics. Although calibration accuracies of less than 1 nm can be achieved in the factory, this requires the use of very expensive spectral lamps and is very laborious so that it cannot be employed as a standard by users. Consequently, the previously used peak search methods do not yield satisfactory results for such low-cost spectrometers.
Furthermore, conventional wavelength calibration methods based on the peak search principle often require spectral lamps or similar light sources which have a large number of peaks that are as narrow as possible. The use of alternative and possibly more cost-effective light sources for wavelength calibration has previously not been possible due to their emission spectra which usually have too few and/or too broad emission peaks. Due to the optical bandwidth of the spectrometers, on the one hand, and the limited number of peaks of spectral lamps on the other hand, only a few suitable peaks are available which limits the quality of the wavelength calibration because all other ranges of the spectrum cannot be used to determine calibration values.